초록 ▼

High throughput VIGS (Virus-Induced Gale Silencing) 기술을 이용하여 식물기능유전체 연구를 수행하였다. 3000개의 Nicotiana benthamiana sequence 풍에서 식물조직발달, 호르몬작용, 신호전달 등에 관련된 유전자 492개와 unknown／hypothetical 유전자 725개 등 총 1217개를 선택하여 pTV00 vector로 cloning하고 Agrobacterium으로 transformation 한 후 각 유전자 당 6개의 Nicotiana benthamiana 식물

High throughput VIGS (Virus-Induced Gale Silencing) 기술을 이용하여 식물기능유전체 연구를 수행하였다. 3000개의 Nicotiana benthamiana sequence 풍에서 식물조직발달, 호르몬작용, 신호전달 등에 관련된 유전자 492개와 unknown／hypothetical 유전자 725개 등 총 1217개를 선택하여 pTV00 vector로 cloning하고 Agrobacterium으로 transformation 한 후 각 유전자 당 6개의 Nicotiana benthamiana 식물체를 사용하여 Agrobacterium infiltration으로 VIGS를 수행하였다. Infiltration후 2-3주 후 식물체의 형질 변화를 control과 비교하여 조사하였다.phenotype은 cell death, leaf yellowing, growth arrest, abnormal organs, premature senescence, flower abnormality, apical dominance breakdown등으로 나누어 분석하였다.1217개 유전자 중 136개 유전자에서 식물체 형질변화가 유도되었다. 나타나는 형질에 대한 결과는 sequence DB 및 유전자 expression profile DB와 연계하여 phenotype DB로 구축하였다. 이 연구를 통하여 식물발달 과정을 콘트롤하는 새로운 유전자들을 발굴하였고 그들 유전자작용 기작을 탐구하므로서 식물발달조절의 전체 네트워크를 이해하고자 하였다. 주요한 유전자의 기능을 여러 분자생물학적, 생화학적, 세포생물학적인 테크닉을 이용하여 심층분석하였다. 이렇게 발굴된 기능성유전자는 고부가가치 형질전환 식물개발에 활용하고자 한다.

Abstract ▼

The purpose of this study is to carry out plant functional genomics using virus-induced gene silencing to understand plant developmental reguatory pathways. We used TRV (tobacco rattle virus)-based VIGS system using Nicotiana benthamiana as a host. Three thousand N. benthamiana cDNAs were random-seq

The purpose of this study is to carry out plant functional genomics using virus-induced gene silencing to understand plant developmental reguatory pathways. We used TRV (tobacco rattle virus)-based VIGS system using Nicotiana benthamiana as a host. Three thousand N. benthamiana cDNAs were random-sequenced, and target genes were selected for VIGS-mediated function analysis. The target genes include plant developmental genes, signal transduction genes, and hormone-related genes. Total 1.217 cDNAs were cloned into pTV00 (VIGS vector), and the recombinant plasmids were transformed into Agrobacterium. Then Agrobacterium suspension was infiltrated into N. benthamiana plants at 3 weeks after germination. After 2-3 weeks after infiltration, the visible phenotypes appeared in the newly emerged plant tissues. Among 1,217 cDNAs, 136 cDNAs showed visible morphological phenotypes, including stunted growth, cell death. changes in leaf color, wrinkled leaves, changes in plant architecture, and flower abnormality. Thus using VIGS, we identified many novel genes that modulate plant developmental processes and characterized their action mechanisms in plant development using various molecular and biochemical tools. We also probed the protein-protein interaction in order to understand the global picture of developmental regulatory networks. The detailed results obtained in this project were summarized below.
We identified NtRpn3, a regulatory subunit of 26S proteasome. as an interacting protein of NtCDPKl calcium-dependent protein kinase in Nicotiana tabacum using yeast two hybrid system. Rpn3 in yeast is an essential protein involved in proteolysis of cell cycle regulatory proteins, and the carrot homologue of Rpn3 was previously isolated as a nuclear antigen that is mainly expressed in the meristem. NtCDPK1 physically interacts with NtRpn3 in vitro in a Ca2＋-independent manner, and phosphorylates NtRpn3 in a Ca2＋-dependent manner wi th Mg2＋ as a cofactor. NtCDPK1 and NtRpn3 are colocalized in the nucleus. nuclear periphery and around plasma membrane in vivo. Both NtCDPK1 and AtRpn3, an NtRpn3 homolog of Arabidopsis, are mainly expressed in the rapidly proliferating tissues including shoot and root meristems, and developing floral buds. Virus-induced gene silencing of either NtRpn3 or NtCDPK1 resulted in the phenotypes of abnormal cell morphology and premature cell death in newly emerged leaves. Finally, NtCDPK1 interacts with NtRpn3 in vivo as shown by co-immunoprecipitation. Based on these results, we propose that NtCDPK1 and NtRpn3 are interacting in a common signal transduction pathway possibly for regulation of cell division, differentiation, and cell death in tobacco.
Proteasomes constitute the major machinery to degrade or process proteins by ATP／ubiquitin-mediated proteolysis. Recent findings suggest a pivotal role of the ubiquitin／proteasome pathway in the regulation of apoptosis in animal cells. Here we show that virus-induced gene silencing of two different subunits of the 26S proteasome, the a6 subunit of the 20S proteasome and RPN9 subunit of 19S regulatory complex, both activated the programmed cell death (PCD) program, accompanied by reduced proteasome activity and accumulation of polyubiquitinated proteins. These results demonstrate that disruption of proteasome function leads to PCD in plant cells. The affected cells showed morphological markers of PCD including nuclear condensation and DNA fragmentation, accompanied by the 10-fold higher production of reactive oxygen species and increased ion leakage for three-fold. Similar to apoptosis in animal system, mitochondrial membrane. potential was decreased, cytochrome c released from mitochondria to cytosol, and caspase 9- and caspase 3-like proteolytic activities detected in the cells. Interestingly, this proteasome-mediated PCD stimulated the expression of only a subset of transcripts that are highly induced during pathogen-mediated HR cell death, indicating that the two PCD pathways are differentially regulated. Taken together, these results provide the first direct evidence that proteasomes playa role in the regulatory program of PCD in plants. Controlled inhibition of proteasome activities may be involved in developmentally- or environmentally-activated plant cell death programs.The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbP4F gene encoding the 6 suhuni t of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsi s, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome -type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems. but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaC1 and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus.
NtFHA1 encodes a novel protein containing the FHA (Forkhead-associated) domain and the acidic domain in Nicotiana tabacum. NtFHAl functions as a transactivator and targeted to the nucleus. The FHA domain of NtFHA1 is significantly homologous in sequences to that of Fh11 forkhead transcription factor of yeast. FHL1 was previously identified as a suppressor of RNA polymerase Ⅲ mutations. and the fh11 deletion mutant exhibited severe growth defects and impaired rRNA processing. Ectopic expression of the FHA domain of NtFHA1, but not its mutant form, resulted in severe growth retardation in yeast. Similarly, expression of Fh11, its FHA domain, or the chimeric Fh11 containing the NtFHA1-FHA domain also inhibited yeast growth. Yeast cells overexpressing the FHA domains of NtFHA1 and Fh11 contained lower levels of mature rRNAs and exhibited rRNA processing defects similarly to the fh11 null mutant. The chimeric Fh11, but not the mutant form with a small deletion in its FHA domain, fully complemented the growth and rRNA processing defects of the fh11 null mutant, demonstrating that the FHA domain of NtFHA1 can functionally substitute for the FHA domain of Fh11.These results demonstrate that the FHA domains of NtFHA1 and Fh11 are conserved in their structure and function, and that the FHA domain of Fh11 is critically involved in regulation of rRNA processing in yeast. NtFHA1 function in plants may be analogous with that of Fh11 in yeast.

목차 Contents

• 제 1 장 연구개발과제의 개요...21
• 제 2 장 국내외 기술개발 현황...23
• 제 3 장 연구개발수행 내용 및 결과...27
• 제 4 장 목표달성도 및 관련분야에의 기여도...109
• 제 5 장 연구개발결과의 활용계획...111
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보...111
• 제 7 장 참고문헌...113
• 특정연구개발사업 연구결과 활용계획서...119